The Primary Study Of Global Acetylation Of Histone In Human Hepatocellular Carcinoma

BackgroundDNA in the eukaryotic genome exists in nucleus as chromatin. Nucleosome, which is formed by core histone protein ( H2A, H2B, H3 and H4 ), H1 and DNA, is the basic unit of eukaryotic chromatin. In the process of gene expression or selective gene silencing, dynamic and large-scale changes occur on the structure of chromatin while the DNA that packaged into chromatin rarely changes in sequence. The changes influence the transcriptional activity at some certain regions of genome. Regulation of gene expression by influencing transcriptional activity that is not accompanied with changes in DNA sequence is defined as epigenetics. It is believed for a long time that gene mutations promote oncogenesis. However, more and more evidences tell us that epigenetics is also important in the initiation and progression of tumor. Epigenetics includes DNA methylation and histone modification. These modifications can change the structure of chromatin and modify transcription. The disturbance of transcription leads to hyperplasy, even causes cancer.Histone modification is more complicated than the DNA methylation, and different histone (histone H3/H4) have different types of modifications, including acetylation, methylation, phosphorylation, ubiquitination, glycosylation, carboxylation and so on. Among these modifications acetylation is the most important one. Histone acetylation is a dynamic, reversible process which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs cause hyperacetylation while HDACs have the opposite effect. Many researches have confirmed that disequilibrium (hyperacetylation/hypoacetylation) of histone acetylation has a close relationship with initiation of tumor. Histone acetylation or deacetylation can change the morphous of cells; On the other hand, HATs and HDACs can influence the differentiation and generation of cells by interacting with oncogenes and anti-oncogenes. In addition, researches have found that post-translational modifications of histone occur mainly on the promoters of genes. In fact, other than being targeted to promoters, modifications of histones, such as acetylation of lysine, also occur over large regions of chromatin including coding regions and non-promoter sequences, which are referred to as global histone modifications. The modification can influence genetic transcription and also determine the global acetylation state of histones in cell and organism.Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in the world. There are more than 500 thousand new cases of HCC each year and 50% cases of these patients are Chinese. HCC is the second leading cause of cancer deaths in our country. Accompanied with the cognition on the importance of epigenetics in the initiation of tumor, epigenetics has become the hot spot in the HCC's research. Some researches have demonstrated that DNA methylation and disturbance of hisone acetylation play an important role in the initiation of HCC. However, up to now, the role of disturbance of hisone acetylation has been studiedonly in vitro by using different chemicals which can change the state of acetylation, and different researches displayed discrepancy results. Some researches reveal that some chemical materials (mainly histone deacetylase inhibitor such as Trichostatin A, diallyl disulfide) which cause histone hyperacetylation can induce apoptosis, cell cycle arrest, antiproliferative effect in HCC. Conversely, some researches exhibit that hypoacetylation (caused by Curcumin, Ni²⁺ and 1, 10-phenanthroline-Cu²⁺) promote the apoptosis of HCC. Contradictory results in vitro spur us to get knowledge about the global acetylation state of histone in HCC. Today, regulating the state of acetylation of histone becomes an important target on cancer treatment with drugs. The knowledge of global acetylation state of histone in HCC will provide evidence for the drug research and clinical application.Materials and Methods1. PatientsA total of 82 patients with primary HCC undergoing hepatectomy or liver transplantation in the Department of Hepatobiliary and Pancreatic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine, were involved in the study. The diagnosis of HCC was confirmed by histopathological examination of each resected tissue. Samples of the HCC and para-cancerous tissue were collected. Collected clinical data such as age, gender, history of Viral Hepatitis Type B, background of cirrhosis, level of AFP, tumor numbers(solitary or mutiple), tumor size, tunicary tumors, portal vein tumor thrombus , TNM grading, histopathological grading of the tumors.2. Microarray chip preparation and immunohistochemistry detectionImmunohistochemistry combined with tissue microarrays was employed to determine the expression of acetylation of histone on HCC tissue and para-canceroustissue. We analysed the levels of acetylated histone H3 (Lys 9) and H4 (Lys 8), using highly specific antibodies (antibody to acetyl-histone H3, acetyl-histone H4).3. Evaluation of stained resultsThe nuclei which are brown stained are regarded as positive cells; For evaluation of expression results a score corresponding to the sum of both staining intensity and percentage of positive cells was determined. Staining intensity: 0, negative; 1, weak; 2, intermediate; 3, strong. Percentage of positive cells: 0, negative ; 1, <25% positive cells ; 2, 25%-50% positive cells ; 3 , 50%-75% positive cells ; 4, >75% positive cells . A score greater than 2 was regarded as positive, while the score lower than or equal to 2 was regarded as negative. The unstained cells might still contain the acetylations at certain genomic loci but their levels are below the detection limits, which are considerably decreased in these cells. The positive results are regarded as a high level global acetylation of histone.4. To compare the level of global acetylation of histone H3/H4 in HCC tissue with that of para-cancerous tissue.5. To analyze whether the level of acetylation of histone H3/H4 correlated with clinical data.6. Statistical analysisx² test and kolmogorov-smirnov test were used for statistical analysis. All analyses were performed with the SPSS statistical package on a personal computer, Release 13.0. All probability values were two-sided, and P≤0.05 was considered significant.Results1. Tissue microarray was successfully constructed and the tissue was fit for the research.2. Among the 82 cases of HCC , the positive rate of acetylation of histone H3/H4 were 68.3% (56/82) 81.7% (67/82), significantly higher than the para-cancerous tissue 31.7% (26/82), 45.1% (37/82) (P<0.05).3. Expression of the two kinds of proteins was not related to clinical characters, including age, gender, history of Viral Hepatitis Type B , background of cirrhosis , level of AFP, tumor numbers(solitary or mutiple), tumor size, tunicary tumors, portal vein tumor thrombus, TNM grading, histopathological grading of the tumors(P>0.05) .ConclusionsGlobal acetylation of histone in HCC tissue is significantly higher man that in the para-cancerous tissue. The result revealed that global hyperacetylation of histone H3/H4 is related to the genesis of HCC. However, the mechanism is not clear yet and should be studied in the future. It needs further research and evaluation for the drug's precise mechanisms, therapeutic effects and clinical applications on the treatment of HCC, which target on regulating the disturbance of acetylation of global histone.